Characterization of the M50/p35 binding site of M53/p38

7.1 Distinct M53-point mutants lost to some extent the ability to localize to M50/p35

Insertions of five amino acids helped to identify a binding motif but the contribution of the inserted amino acids that differ between the different insertion mutants is difficult to evaluate. In order to define the relative role of individual amino acids with respect to M50/p35 binding the 12 amino acids which are conserved in beta-herpesviruses within the N-terminal half of CR1 (aa 112-137) were one by one replaced by alanine. After transfection of NIH 3T3 cells with the M53/p38 point mutants alone or co-transfection with M50/p35, respectively, the sub-cellular localization of the M53/p38 mutants was analyzed.

All M53/p38 point mutants showed a diffuse nuclear staining when expressed alone (Fig. 26, first vertical row). After co-expression with M50/p35, the intra-nuclear distribution of M53 point mutants fell into three classes of phenotypes:

i. Mutants that redistributed to the nuclear rim and recruited M50/p35 similar to wt M53/p38 (M53P123A, M53D124A, and M53E126A).

ii. Mutants that had apparently lost the ability to redistribute to the nuclear rim in presence of M50/p35 (M53Y129A (Fig. 26), M53L130A and M53I133A).

iii. Mutants with intermediate phenotypes. Several mutants fell into this class and their phenotypic appearance was variable. One subset showed abundant diffuse intra-nuclear staining in addition to co-localization with M50/p35 at the nuclear rim (M53L112A (Fig. 26), M53H116A, M53F119A, M53I137A). The second type of mutants co-localized with M50/p35 at least to some extent.

D. Results

Figure 26. Functional analysis of M53 point mutants. Sub-cellular localization of M53 point mutants. NIH 3T3 cells were transfected alone with M53 point mutants L112A, L125A, K128A and Y129A (first vertical row) or co-transfected with wt M50/p35 (second to fourth vertical rows).

Single transfected cells were stained with rat specific antiserum against M53/p38. For detection a fluorescein-conjugated secondary antibody was used (green). Co-transfected cells were treated as described above and co-stained with an M50/p35 specific rabbit serum, which was detected by Texas red coupled secondary antibody (red).

D. Results The remarkable phenotype, however, was a new distribution of M53/p38 mutants upon co-expression of M50/p35 resulting in the formation of discrete intra-nuclear aggregates (M53L125A) and fibrous structures (M53K128A, Fig.

26) which did not co-localize with M50/p35. In the absence of M50/p35 the intra-nuclear distribution of the mutants was indistinguishable from that of wt M53/p38.

7.2 Distinct M53/p38 point mutants showed reduced binding to M50/p35 and resulting viruses were strongly attenuated

Next, 293 cells were co-transfected with M50ST and the set of alanine scanning M53/p38 mutants. Strep-Tactin Sepharose pull-down was performed to analyze the binding capacity of the M53 point mutants to M50ST. Remarkably, despite the different distribution phenotypes, all M53 point mutants could still be retrieved by M50ST pull-down, including those that did not show a detectable interaction in the co-localization assay (Fig. 27A). Notably, the binding was found significantly reduced for the mutants M53L112A, M53L125A, M53Y129A and M53M133A.

A

Figure 27A. Pull down analysis of 12 M53 point mutants with Strep-tagged M50/p35. 293 cells were co-transfected with the indicated M53 point mutants and M50ST. Total cell lysates were analyzed to test the protein expression by Western blot using specific antiserum against M53/p38 (upper panel, T). Proteins complexed with M50ST were precipitated by Strep-Tactin Sepharose. Desthiobiotin eluates were analyzed by SDS-PAGE and blotted on membranes.

Signals for M53 point mutants were visualized by Western blot with M53/p38 specific antiserum (lower panel, B). As positive control for precipitation functional insertion mutant M53i104 was used (lane 1) and M53 insertion mutant M53i128 served as negative control (lane 2).

D. Results Since there was a substantial degree of variability in the co-localization assay and certain heterogeneity of interactions detectable by the pull-down assay, we tested the M53 point mutants for functionality in the viral context. Interestingly, the null phenotype of ∆M53FRT-BAC could be reverted by all M53 point mutants. Usually about 11 days are required to detect the first virus plaques after transfection of the ectopically complemented deletion genome.

Remarkably, virus reconstitution time and plaque formation was significantly delayed to three weeks for mutants M53K128A and M53L130A. Virus reconstitution from the BAC carrying M53Y129A required even six weeks.

B

Figure 27B. Rescue of ∆M53-BAC by M53 point mutants K128A, Y129A and L130A. Growth kinetics of the viruses derived from pM53E or pM53K128AE, pM53Y129AE and pM53L130AE.

NIH 3T3 cells were infected with the respective viruses. Supernatants of the infected cells were harvested on the indicated days, and virus titers were determined by plaque assay.

Accordingly, the growth of mutants M53K128A and M53L130A was reduced by one order and M53Y129A was attenuated more than 3 orders of magnitude in comparison with a genome complemented by wt M53 ORF (Fig. 27B). These data show that already the exchange of a single aa within the binding region of M53/p38 can strongly affect M50/p35 binding but cannot completely abolish a functional M50/p35-M53/p38 interaction.

D. Results 7.3 Exchange of only two aminoacids abolishes M50-M53 interaction and rescue of the M53 null phenotype

To rigorously test the binding site we replaced either two adjacent critical amino acids (M53YL129-130A) or three adjacent amino acids by alanine (M53KYL128-130A). In addition, we deleted the sequences aa 108-136 of M53 (M53∆106-136). As expected, each of these mutants completely failed to co-localize with M50/p35 (Fig. 28A), failed to interact with M50ST in the pull-down assay (Fig. 28B) and also failed to rescue the M53 null phenotype.

A

D. Results

Figure 28A. Functional analysis of M53 point-and deletion mutants. Sub-cellular localization of M53 point-and deletion mutants. NIH 3T3 cells were transfected alone with M53 point- and deletion mutants M53YL129-130A, M53KYL128-130A, ∆16-136A, ∆16-106N and

∆16-136N (first vertical row) or co-transfected with wt M50/p35 (second to fourth vertical rows).

Single transfected cells were stained with rat specific antiserum against M53/p38. For detection a fluorescein-conjugated secondary antibody was used (green). Co-transfected cells were treated as described above and co-stained with an M50/p35 specific rabbit serum, which was detected by Texas red coupled secondary antibody (red).

B C

Figure 28B and C. Analysis of the interaction of M50/p35 with M53 point- and N-terminal deletion mutants. (B) Pull down analysis of M53 point mutants YL129,130A, KYL 128-130A and M53-∆108-136 with Strep-tagged M50. 293 cells were co-transfected with M53 point mutants and M50ST. Expression of the constructs was tested by SDS-PAGE of total cell lysates and Western blot using specific M53/p38 antiserum (upper panel, T). Proteins complexed with M50ST were precipitated by Strep-Tactin Sepharose. Desthiobiotin eluates were analyzed by SDS-PAGE and blotted on membranes. Signals for M53 point mutants were visualized by Western blot with M53/p38 specific antiserum (lower panel, B). As a positive control a functional insertion mutant, M53i104, was used (lane 1) and M53i128, which failed to bind to M50/p35 served as negative control (lane 2). (C) Pull down analysis of N-terminal deletion mutants of M53/p38. 293 cells were co-transfected with M53 N-terminal deletion mutants M53-∆16-106NLS or M53-∆16-136NLS and M50ST. Analysis of total cell lysates (upper panel, T) and protein complex formation (lower panel, B) was performed as described above.

The N-terminal deletion mutant of M53-∆16-106NLS carrying deletions of aa 16 to aa 106 and an artificial NLS rescued the M53 deletion after ectopic re-insertion into the mutant MCMV-BAC indicating that this mutant can interact with the M50/p35. To confirm the M50/p35 interaction site mapping data, the N-terminal deletion was increased to aa 16 to aa 136 (M53-∆16-136NLS). As

D. Results expected, the M53-∆16-106NLS did interact with M50ST in the pull-down assay whereas 136NLS did not (Fig. 28C). Also, in contrast to M53-∆16-106NLS the N-terminal deletion mutant M53-∆16-136NLS was not able to rescue the M53 null phenotype upon insertion into the deletion BAC.